I propose to study the genetic control of the variable (V) regions of mouse antibodies specific for a particular antigen by direct chemical analysis of V region genes. Antibody V genes will be isolated from DNA of undifferentiated cells of different inbred strains of mice and from B cells at different stages of differentiation. We have already established hybrid cell lines obtained by fusion of myeloma cells with spleen cells from hapten protein conjugate primed mice. These hybrid cell lines secrete large amounts of antihapten antibodies and are probably derived from plasma B cells which were arrested at that stage of differentiation and fused with myeloma cells. Specific B cells at various stages of differentiation, namely pre-B and resting lymphocytes, will be obtained from fetal liver and bone marrow of adult mice. These will be made immortal by the somatic cell hybridization technique. Several rearranged V region genes will be prepared from a family of mouse hybridoma cell lines producing antibodies of similar antigen specificity and expressing a characteristic idiotype. Special emphasis will be put on rearranged VH genes which will be isolated by using a specific JH probe. The rearranged DNA sequences will be cloned by recombinant DNA techniques and will be characterized by restriction enzyme mapping and DNA sequencing. VH germ line genes will be identified and cloned by using specific unique probes obtained from cloned rearranged VH genes. The objective of this study is to determine the genetic basis for inheritable phenotypic antibody heterogeneity observed in the immune response of some inbred strains of mice to a particular antigen, the hapten 4(hydroxy-3 nitrophenyl)acetyl. We will ask whether heterogeneity is due to somatic mutation and somatic rearrangement of a single VH germ line gene, or due to the expression of several related germ line genes which undergo somatic mutation and somatic rearrangements. The analysis of several somatic variants of the same germ line gene will allow us to distinguish between random and site specific somatic mutation. Our studies will involve analysis of rearranged VH genes obtained from DNA of B cells at different stages of differentiation. The comparative studies of rearranged VH genes obtained from DNA of antigen-stimulated plasma B cells and B cells at early stages of differentiation will give us an idea of how much somatic mutation occurs at these different stages of B cell differentiation.